Chitosan-coated wires for biosensing

ABSTRACT

A method of forming a bioelectronic device including a protein on an electrically conductive substrate, by electrodepositing aminopolysaccharide chitosan on the substrate while applying a cathodic voltage to the substrate, to form an aminopolysaccharide chitosan film thereon, applying an anodic voltage to the substrate in the presence of NaCl to activate the aminopolysaccharide chitosan film so that it is reactive with protein. The method also optionally includes reacting the aminopolysaccharide film, after activation thereof, with the protein, so that the protein assembles on and is coupled to the substrate, thereby forming a bioelectronic device. The protein can include single or multiple protein species, and including biosensing proteins. Additional methods include biosensing of electrochemically active compounds either present in a sample or generated during a biological recognition event and devices useful in such methods. The resulting devices are useful as sensors in hand-held devices, textiles, garments and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under theprovisions of 35 U.S.C. §371 of International Patent Application No.PCT/US09/59372 filed Oct. 2, 2009, and published on Apr. 8, 2010 asInternational Publication No. WO 2010/040047, which in turn claims thebenefit of U.S. Provisional Patent Application No. 61/102,009 filed Oct.2, 2008 in the names of Xiao-Wen Shi, Yi Liu and Gregory F. Payne for“Electrical Signal Guided Protein Assembly” and further claims thebenefit of U.S. Provisional Patent Application No. 61/238,277 filed Aug.31, 2009 in the names of Yi Liu, Xiao-Wen Shi, Gregory F. Payne and W.Lee Meyer for “Chitosan-Coated Wires for Biosensing.” The disclosures ofsuch international patent application and U.S. priority patentapplication are hereby incorporated herein by reference in theirrespective entireties, for all purposes.

GOVERNMENT RIGHTS IN INVENTION

This invention was made with government support under Grant No.CBET-0650650 and Grant No. EFRI-0735987, awarded by the National ScienceFoundation and Grant No. W91B9480520121 awarded by the Department ofDefense. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a system and method for utilizingelectrical signals to effect protein assembly, and devices producedthereby. Particularly, methods of making and using devices containingchitosan-coated electrodes are provided. The devices produced by themethods described herein provide convenient biosensing platforms thatcouple biological capabilities for selective detection with electronicstechnology for signal transduction.

DESCRIPTION OF THE RELATED ART

Electronics and biology provide unique capabilities for sensing, andthese capabilities are increasingly being employed for analysis outsidethe laboratory. Electronics offer an array of sensors to detect physicaland mechanical conditions, and electronics offer extensive signalprocessing capabilities. Recent investigations aim to incorporate thesensing and transduction capabilities of electronics into wearableinstrumentation (Junker, H. et al., PatternRecognit.2008, 41,2010-2024.) or smart textiles. (Coyle, S., et al. MRSBull.2007, 32,434-442; Abouraddy, A. F., et al. Nat.Mater. 2007, 6, 336-47; Hamedi,M., et al. Nat.Mater.2007, 6, 357-62.) These smart textiles can monitorthe wearer's actions, wirelessly communicate this information, andpotentially even guide motion. Smart textiles that offer such physicaland mechanical sensing capabilities are expected to provide importantbenefits (e.g., to detect that a house-bound person has fallen or toassess danger to a firefighter).(Huang, C. T., et al.Sens.Actuator,2008, 141, 396-403; Jung, S., et al.SmartMater.Struct.2006, 15, 1872-1876; Devaux, E., et al.,Trans.Inst.Meas.Control 2007, 29, 355-376; Liu, Y. Y., et al.J.Mater.Chem. 2008, 18, 3454-3460.) Such textiles would be even smarterif they could detect and report chemical and biochemical information.(Wallace, G., et al. SoftMatter 2007, 3, 665-671.)

Biological recognition elements (e.g., nucleic acids, enzymes andantibodies) permit the selective detection of chemical and biochemicalinformation, and these elements are extensively employed for laboratoryanalysis (e.g., microarrays and immunoassays). There is a growinginterest in creating platforms that enable these biological recognitionelements to be employed outside the laboratory. Several groups areadvocating the use of fibers and fabrics as platforms for biosensing inthe field. For instance, fiber optics are commonly considered forchemical and biochemical sensing because optical fibers offerconsiderable signal transduction capabilities. (Wolfbeis, O. S., et al.Anal.Chem.2008, 80, 4269-83; Leung, A., et al. Sens.Actuators, 2007,125, 688-703; Leung, A., et al. Sens.Actuators, 2008, 131, 640-645; Ko,S. H., et al. Biosens.Bioelectron. 2006, 21, 1283-1290; Rijal, K., etal. Biosens.Bioelectron. 2005, 21, 871-880; Epstein, J. R., et al.Chem.Soc.Rev. 2003, 32, 203-14.) One limitation of fiber optic systemsis that the incorporation of the biological sensing element is notalways simple or benign. Alternatively, polymer based fibers (andnanofibers) and fabrics (Schiffman, J. D., et al. Biomacromolecules2007, 8, 594-601; Schiffman, J. D., et al. Polym.Rev. 2008, 48, 317-352;Klossner, R. R., et al. Biomacromolecules 2008, 9, 2947-53; Desai, K.,et al. Biomacromolecules 2008, 9, 1000-6; Kriegel, C., et al.Crit.ReV.FoodSci.Nutr. 2008, 48, 775-797) are being examined as aplatform for biosensing. (Shi, X. W., et al. Biomacromolecules 2008, 9,1417-23; Shi, Q., et al. Biomaterials 2008, 29, 1118-1126). In manycases, polymeric fibers are more readily biofunctionalized althoughsignal transduction with polymers is not as straightforward whencompared to optical fibers.

There is great interest in coupling the capabilities of electronics withthe molecular recognition properties of biology to generate hand-helddevices that can diagnose diseases at the point-of-care, analyzeenvironmental samples in the field, and assess food safety from the farmto the table. A key challenge is the integration of the labilebiological recognition elements (e.g., proteins) at specific deviceaddresses or locations.

There therefore remains a need in the art for development of abiosensing platform that can be readily biofunctionalized and thatallows the transduction of biological recognition into convenientelectrical signals. The present invention provides such a platform andmethods of making and using the same.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method of forming abioelectronic device including a protein on an electrically conductivesubstrate, comprising:

-   -   electrodepositing aminopolysaccharide chitosan on the substrate        while applying a cathodic voltage to the substrate, to form an        aminopolysaccharide chitosan film thereon;    -   applying an anodic voltage to the substrate in the presence of        NaCl to activate the aminopolysaccharide chitosan film so that        it is reactive with protein; and    -   reacting the aminopolysaccharide film, after activation thereof,        with the protein, so that the protein assembles on and is        coupled to the substrate, thereby forming said bioelectronic        device.

Another aspect of the invention relates to a method of forming anelectrically conductive substrate adapted for assembly of proteinspecies thereon, said method comprising:

-   -   electrodepositing aminopolysaccharide chitosan on the substrate        while applying a cathodic voltage to the substrate, to form an        aminopolysaccharide chitosan film thereon; and    -   applying an anodic voltage to the substrate in the presence of        NaCl to activate the aminopolysaccharide chitosan film so that        it is reactive with protein.

The invention relates in a further aspect to a bioelectronic device,e.g., a hand-held device, comprising:

-   -   an electrically conductive substrate;    -   an electrodeposited aminopolysaccharide chitosan film on the        substrate; and    -   protein conjugated to the electrodeposited aminopolysaccharide        chitosan film.

The invention relates in a further aspect to a bioelectronic precursordevice, comprising:

-   -   an electrically conductive substrate; and    -   an electrodeposited aminopolysaccharide chitosan film on the        substrate, wherein said film is adapted to react with and        conjugate a protein.

In another aspect the invention relates to a method of detecting thepresence of a protein in a sample comprising the steps of:

-   -   contacting the sample with a bioelectronic device,        comprising: i) an electrically conductive substrate; and ii) an        electrodeposited aminopolysaccharide chitosan film on the        substrate, wherein said film is reactive with a protein under        conditions sufficient to react the film with the protein;    -   detecting the presence or absence of the reacted protein.

In a still further aspect the invention relates to a method oftransducing enzyme substrate recognition into an electrical signalcomprising the steps of:

-   -   obtaining a bioelectronic device, comprising: i) an electrically        conductive substrate; and ii) an electrodeposited        aminopolysaccharide chitosan film on the substrate, wherein said        film is conjugated with an enzyme;    -   contacting the bioelectronic device with a substrate-containing        sample, wherein the substrate is reactive with the enzyme of the        bioelectronic device under conditions sufficient to react the        substrate and the enzyme;    -   detecting the anodic current, wherein a change in anodic current        is an electrical signal indicative of enzyme substrate        recognition.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the scheme of the invention, where goldwires are biofunctionalized using cathodic signals to electrodepositchitosan and anodic signals to activate the chitosan for proteinassembly.

FIG. 2 relates to electro-addressing of proteins, including (a) aschematic illustration of a two-step approach in which a cathodic signalis used to electrodeposit an aminopolysaccharide chitosan, and an anodicsignal is used to electrochemically activate the chitosan film forprotein assembly; (b) a chip with two electrically-independent goldelectrodes (1 mm×8 mm) patterned onto silicon; and (c) high-resolutionXPS spectra of a control chitosan film and a chitosan film oxidized at0.9 V to achieve a charge transfer of 80 C/m².

FIG. 3 relates to assembly of target proteins at individual electrodeaddresses, including: (a) a chip with 6 electrically independentelectrode addresses (250 μm wide gold lines spaced 250 μm apart); (b) aschematic of avidin assembly to electrochemically-activated chitosanfilms and a fluorescence photomicrograph showing binding of labeledbiotin; (c) a schematic of Protein G assembly toelectrochemically-activated chitosan films and a fluorescencephotomicrograph showing binding of labeled human IgG; and (d) acorrelation between protein assembly (as determined from image analysisof fluorescence) and the extent of chitosan film activation (as measuredby the charge transfer, Q).

FIG. 4 relates to multiplex protein assembly, including: (a) a schematicillustration of the assembly of Green Florescent Protein (GFP) on theleft-most electrodes and Red Florescent Protein (RFP) on the right-mostelectrodes; (b) a fluorescence photomicrograph of the two proteinsassembled on the chip, wherein the top two images were obtained using asingle filter while the bottom image is a composite using both red andgreen filters, with image analysis performed for the composite image;and (c) plots of fluorescence intensity versus charge transfer (Q) forassembly of the two proteins, with an independent calibration indicatingthat the assembled protein is on the order of 10-100 pmol/cm².

FIG. 5 relates to NaCl being required for chitosan film activation, with(a) a chip with 2 electrically-independent electrode addresses; (b)protein assembly onto electrodeposited chitosan film, with the controlchitosan film on the left electrode anodically activated in 0.1 Mphosphate buffer (pH=7.0), while chitosan film on the right electrodewas activated in 0.1 M phosphate buffer containing 0.1 M NaCl, and afteractivation, the chip was immersed in a solution containing the model RedFluorescent Protein (RFP), and (c) analysis of the image of (b).

FIG. 6 relates to chitosan being required for electrochemical proteinassembly, including: (a) chitosan electrodeposited on the left electrodeof the chip of FIG. 5( a) while the right electrode was untreated, andafter anodic oxidation of both electrodes (0.9 V, Q=80 C/m²), the chipwas immersed in a solution of labeled bovine serum albumin (BSA), withthe fluorescence photomicrograph indicating that the labeled BSA wasassembled on the chitosan-coated (left) electrode but not onto the baregold electrode; and (b) analysis of the image in FIG. 6( a).

FIG. 7 relates to activated chitosan films, as “de-activated” bytreatment with a reducing agent, wherein chitosan was electrodepositedon both electrodes of the chip of FIG. 5( a), the right electrodeanodically activated (0.9 V, Q=80 C/m²) and thereafter was treated withNaBH₄ (0.2 mg/ml) for 1 hr, following which the left electrode wasanodically activated, and the chip was immersed in a solution containinglabeled BSA, the fluorescence photomicrograph indicating that treatmentby the reducing agent disrupts protein assembly; and (b) analysis of theimage of FIG. 7( a).

FIG. 8 relates to electrodeposition of fluorescently labeled chitosanonto gold wires. (a) Schematic illustrating chitosan electrodepositiononto a gold wire in response to cathodic signals. (b) Fluorescencephotomicrographs of labeled-chitosan deposited onto gold wires.Deposition was achieved by applying a cathodic voltage at a constantcurrent density (12.6 A/m²) for 15, 45, and 90 s. The control is goldwire immersed in chitosan solution for 90 s in the absence of an appliedvoltage.

FIG. 9 relates to electrochemical transduction with chitosan-coatedwire. (a) Schematic illustrating that phenol (i.e., catechin) can beelectrochemically oxidized to generate a detectable electrical signal(current). (b) Cyclic voltammograms (CVs) for a 2 mM catechin solution(0.1 M phosphate buffer, pH) 7.4) show that chitosan-coated gold wirehas a strong anodic peak. A smaller anodic peak is observed for anuncoated gold wire in the presence of 2 mM catechin. A control is achitosan-coated gold wire immersed in buffer (without catechin). Scanrate) 0.1 V/s.

FIG. 10 relates to quantitative analysis of catechin withchitosan-coated wires. (a) Schematic of the i-t(current vs time)measurements using a standard three-electrode configuration. Electrodeswere immersed in a buffered solution (0.1 M phosphate, pH 7.4), and theworking electrode was biased to 0.5 V. (b) Correlation between thecurrent and catechin level. Inset shows current steps upon sequentialadditions of 0.6 μM catechin. (c) Sensitivity of electrochemicaldetection of catechin is increased by increasing the number ofchitosan-coated wires.

FIG. 11 relates to catechin oxidation confers fluorescence tochitosan-coated gold wires. Fluorescence images and image analysis ofchitosancoated wires show a progressive increase in fluorescence withcatechin level in the solution. The wires were immersed in solutionscontaining different concentrations of catechin (0, 0.2, 0.5, and 1.0mM) and biased to an anodic voltage of 0.5 V for 2 s. The control is achitosan-coated wire that was immersed in a 1.0 mM catechin for 2 s inthe absence of an applied voltage.

FIG. 12 relates to electrochemical protein conjugation ontochitosan-coated wires. (a) Fluorescence images and image analysisindicate a progressive increase in the conjugation of red fluorescentprotein (RFP) with anodic activation time. Electrochemical conjugationwas achieved by immersing chitosan-coated wires in a 3 μM RFP solution(0.1 M phosphate, 0.1 M NaCl, pH 7.4) and applying a 0.9 V anodicvoltage. The control is a chitosan-coated wire that was immersed in thesame RFP solution for 120 s (no voltage applied). (b) Correlationbetween RFP assembly (as determined from image analysis of fluorescence)and the extent of chitosan film activation (as measured by the totalcharge transferred).

FIG. 13 relates to cyclic voltammograms for chitosan-coated wiresfunctionalized with GOx. (a) Wires biofunctionalized by codepositionshow a strong anodic signal in a 2 mM glucose solution. Chitosan and GOxwere codeposited from a solution (1% chitosan, 680 U/mL GOx, pH 5.6) ata current density of 12.6 A/m² for 20 s. (b) Wires biofunctionalized byelectrochemical conjugation show a strong anodic signal in a 2 mMglucose solution. GOx was electrochemically conjugated by immersing thechitosan-coated wires in a solution (0.1 M phosphate, 0.1 M NaCl, 680U/mL GOx, pH 7.4) and applying an anodic voltage of 0.9 V for 60 s. As acontrol, electrochemical conjugation was performed with nonfat dry milk(5% milk), and the milk-conjugated chitosan-coated wire was tested inthe 2 mM glucose solution.

FIG. 14 relates to quantitative signal transduction fromGOx-functionalized chitosan-coated wires. GOx was electrochemicallyconjugated to chitosan-coated wire and glucose was detectedamperometrically (as in FIG. 10). (a) Standard curve between anodiccurrent and glucose concentration at low concentrations (each aliquotincreased the glucose concentration by 0.25 mM). (b) Standard curve overa broader concentration range using a different GOx-functionalizedchitosancoated wire (each aliquot increased the glucose concentration by1.0 mM). Note the data in (a) are reproduced in (b), open circles.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS THEREOF

The present invention is based on the discovery of a simple, safe andgeneric approach for assembling proteins in response toelectrode-imposed electrical signals and for biofunctionalization of theinitial platform. This approach relies on the aminopolysaccharidechitosan that can be electrodeposited in response to cathodic signalsand then electrochemically activated by anodic signals. Theelectodeposited and electroactivated chitosan films react with proteinsto assemble them with spatial-selectivity and quantitative-control. Thepresent inventors have found that the assembled proteins retain theirnative structure and biological functions. The resulting devices areuseful in methods of biosensing using the conjugated proteins.Furthermore, the present invention provides additional devices for andmethods of biosensing, where the biosensing platforms contain elementspermeable to small molecules, allowing detection of electrochemicallyactive compounds, whether present in the solution or generated during abiological recognition event, such as enzyme substrate recognition.

The inventive methodology for on-demand biofunctionalization ofindividual electrode addresses, as discussed more fully hereinafter,provides a reliable capability for assembling proteins for multiplexedanalysis.

In one aspect, the invention relates to a method of forming abioelectronic device including a protein on an electrically conductivesubstrate, comprising:

-   -   electrodepositing aminopolysaccharide chitosan on the substrate        while applying a cathodic voltage to the substrate, to form an        aminopolysaccharide chitosan film thereon;    -   applying an anodic voltage to the substrate in the presence of        NaCl to activate the aminopolysaccharide chitosan film so that        it is reactive with protein; and    -   reacting the aminopolysaccharide film, after activation thereof,        with the protein, so that the protein assembles on and is        coupled to the substrate, thereby forming said bioelectronic        device.

In a specific embodiment, the substrate comprises a multiplexing chiphaving a multiplicity of electrode addresses, wherein the electrodeaddresses independently have protein assembled thereon and coupledthereto. The multiplicity of electrode addresses can have differentprotein species assembled thereon and coupled thereto. The protein cancomprise one or more types of biosensor protein species.

Another aspect of the invention relates to a method of forming anelectrically conductive substrate adapted for assembly of proteinspecies thereon, said method comprising:

-   -   electrodepositing aminopolysaccharide chitosan on the substrate        while applying a cathodic voltage to the substrate, to form an        aminopolysaccharide chitosan film thereon; and    -   applying an anodic voltage to the substrate in the presence of        NaCl to activate the aminopolysaccharide chitosan film so that        it is reactive with protein.

The invention relates in a further aspect to a bioelectronic device,e.g., a hand-held device, comprising:

-   -   an electrically conductive substrate;    -   an electrodeposited aminopolysaccharide chitosan film on the        substrate; and    -   protein conjugated to the electrodeposited aminopolysaccharide        chitosan film.

The invention relates in a further aspect to a bioelectronic precursordevice, comprising:

-   -   an electrically conductive substrate; and    -   an electrodeposited aminopolysaccharide chitosan film on the        substrate, wherein said film is adapted to react with and        conjugate a protein.

The invention in one aspect relates to a two-step approach for enlistingelectrical signals for protein assembly. This approach is illustrated inFIG. 2( a) and relies on the unique physical and chemical properties ofthe aminopolysaccharide chitosan.

In another aspect the invention relates to a method of detecting thepresence of a protein in a sample comprising the steps of:

-   -   contacting the sample with a bioelectronic device,        comprising: i) an electrically conductive substrate; and ii) an        electrodeposited aminopolysaccharide chitosan film on the        substrate, wherein said film is reactive with a protein under        conditions sufficient to react the film with the protein;    -   detecting the presence or absence of the reacted protein.

In a still further aspect the invention relates to a method oftransducing enzyme substrate recognition into an electrical signalcomprising the steps of:

-   -   obtaining a bioelectronic device, comprising: i) an electrically        conductive substrate; and ii) an electrodeposited        aminopolysaccharide chitosan film on the substrate, wherein said        film is conjugated with an enzyme;    -   contacting the bioelectronic device with a substrate-containing        sample, wherein the substrate is reactive with the enzyme of the        bioelectronic device under conditions sufficient to react the        substrate and the enzyme;    -   detecting the anodic current, wherein a change in anodic current        is an electrical signal indicative of enzyme substrate        recognition.

Chitosan is a unique interface material that allows conducting wires tobe biofunctionalized through simple electrical signals. Specifically, inthe claimed invention, cathodic signals are used to direct chitosan toelectrodeposit onto gold wires and anodic signals are used to conjugateproteins to the chitosan-coated wire. In addition, the chitosan-coatingis permeable to small molecules which allows for the electricaldetection of electrochemically-active compounds that are either presentin the external environment or generated by a biofunctionalized chitosancoating. The capabilities for biofunctionalization and transduction areexemplified herein by the detection of glucose by chitosan-coated wiresfunctionalized with the enzyme glucose oxidase. Chitosan-coated wires(or alternatively conducting chitosan fibers) are a simple platform thatmay permit multiplexed biosensing outside the laboratory.

In the methods and devices of the invention, the electrically conductivesubstrate may comprise an electrode, e.g., a gold electrode, on asilicon chip. The electrically conductive substrate of the invention maycomprise any noble or non-noble metal, including, but not limited to:gold, copper, ruthenium, rhodium, palladium, silver, osmium, iridium,and platinum. In another aspect the electrically conductive substrate ofthe invention comprises graphene. In various aspects, the electricallyconductive substrate of the invention is in any form effective forconjugation, as described. Such forms include, but are not limited to awire, a panel, mesh, a scaffold, and the like.

The protein conjugated to the chitosan film on the substrate can be ofany suitable type, such as avidin, an immunoglobulin-binding proteinsuch as protein G, RFP, GFP, or other suitable protein(s).

The substrate may comprise a multiplexing chip having a multiplicity ofelectrode addresses, wherein the electrode addresses independently haveprotein conjugated thereto, e.g., different protein species includingbiosensor protein species.

Electrodeposition of Chitosan

As illustrated in FIG. 1, the present invention provides use of theamino polysaccharide chitosan to serve as the interface between theprotein-based recognition element and a metal wire. Importantly chitosanallows proteins to be assembled in response to imposed electricalsignals without the need for reactive reagents or harsh conditions.(Shi, X. W., et al. Adv.Mater. 2009, 21, 984-988.) Thus,biofunctionalization is simple, safe, and rapid. Further, thechitosan-coating is permeable to small molecules and allows thedetection of electrochemically active compounds that are either presentin the solution or generated during the biological recognition event.Thus, chitosan-coated electrodes can transduce chemical and biologicalinformation into convenient electrical signals.

Chitosan electrodeposition has been extensively studied on planarelectrode surfaces (Wu, L. Q., et al. Langmuir 2002, 18, 8620-8625; Wu,L. -Q., et al. Langmuir 2003, 19, 519-524; Yi, H., et al.Biomacromolecules 2005, 6, 2881-94; Casagrande, T., et al.Mater.Chem.Phys. 2008, 111, 42-49; Pang, X., et al. Surf Coat.Technol.2008, 202, 3815-3821; Bardetsky, D., et al. Surf.Eng. 2005, 21, 125-130;Hao, C., et al. Anal.Chem. 2007, 79, 4442-7) and chitosanelectrodeposition on wires and wire meshes has been demonstrated. (Pang,X., et al. Mater.Chem.Phys. 2005, 94, 245-251; Grandfield, K., et al.Mater.Charact. 2008, 59, 61-67; Pang, X., et al. Mater.Charact. 2007,58, 339-348.)

In a first step of generating devices of the invention, chitosan iselectrodeposited as a thin film onto a cathode surface. Mechanistically,cathodic reactions create a localized region of high pH that induceselectrodeposition due to chitosan's pH-responsive film-formingproperties. Mechanistically, chitosan electrodeposition occurs becausethe locally high pH at the cathode surface induces a sol-gel transitionof the pH-responsive chitosan (chitosan is soluble at pH below it spKaof 6.0-6.5). Once deposited and rinsed, the chitosan film is stable onthe electrode in the absence of an applied potential provided the pH isretained above about 6.3(however, the film re-dissolves at lower pHs).

The second step of generating devices of the invention compriseselectrochemical activation of the deposited chitosan film. Activation isachieved by biasing the underlying electrode to an anodic voltage togenerate a diffusible mediator that can partially oxidize chitosan togenerate substituents on this polysaccharide that are reactive towardproteins.

Electrodeposition and activation of chitosan films for protein assemblycan be carried out under any suitable conditions, as will be readilydeterminable for specific implementations of the invention, based on thedisclosure herein. For example, chitosan can be initiallyelectrodeposited onto gold electrodes patterned onto silicon chips, asshown in FIG. 2( b).

Electrodeposition in an illustrative embodiment can be achieved bypartially immersing the chips in a chitosan solution (e.g., 0.9%chitosan, at a pH of 5.6) and applying a cathodic voltage (for example,at 4 A/m² for 15 seconds) to the specific electrode.

The schematic in FIG. 8( a) provides a further illustrative embodimentwhere electrodeposition is performed by partially immersing the wires ina chitosan solution (1.0% chitosan; pH of 5.6) and applying a cathodicvoltage to achieve a constant current density (12.6 A/m²) for a shorttime (15-90 s). To facilitate visualization, deposition was performedusing fluorescein-labeled chitosan. (Example 6) After deposition, thewires were rinsed and imaged using a fluorescence microscope. Thecontrol wire shown at the left in FIG. 8( b) was immersed in thelabeled-chitosan solution for 90 s, however, no voltage was applied. Theimage and image analysis for this control show little fluorescence,indicating that nonspecific adhesion of chitosan to the gold wire isminimal. The fluorescence on the remaining wires was observed toincrease as the deposition time was increased. These results confirmthat chitosan electrodeposition can be performed on gold wires. Thepresent inventors also observed that chitosan can be effectivelyelectrodeposited onto copper wires.

Protein Deposition

Chitosan films can be activated for protein assembly by anodic oxidationof the underlying electrode in the presence of NaCl. (Shi, X.-W., et al.Adv.Mater. 2009, 21, 984-988.) It has been found that protein assemblydoes not occur if anodic activation is performed in the absence of NaCl.(Example 3) Such result is consistent with chitosan activation by HOClor OCl⁻ species.

In an illustrative embodiment, after electrodeposition of chitosan, thechips are rinsed and the chitosan films are chemically activated byimmersing them in phosphate buffer (0.1 M, pH 7) containing NaCl (0.1M), and biasing the underlying electrode to an anodic potential of (0.9V) for short times (<1 min). After rinsing the chips with the activatedchitosan films, target proteins are assembled by incubating with theprotein-containing solutions for 0.5-1 hr.

It has also been empirically determined that proteins do not assemble toactivated chitosan films that are treated with the reducing agent NaBH₄(0.2 mg/ml) for 1 hr prior to protein assembly. This finding isconsistent with the presence of aldehydes as activated substituents onthe chitosan films.

Direct spectroscopic evidence that anodic oxidation chemically altersthe chitosan films is provided by X-ray photoelectron spectroscopy(XPS). This was analytically shown by first electrodepositing chitosanfilms onto two electrode addresses of the chip shown in FIG. 2( b). Thechitosan film on one electrode was anodically oxidized by biasing theunderlying electrode (0.9 V) to achieve a charge transfer of 80 C/m²(approximately 10 sec). The second electrode served as an unoxidizedcontrol. After activation, the chip was immersed in 0.1 M HCl for 30 minto partially dissolve unreacted chitosan and expose regions of the filmsthat were closer to the electrode surface. The films were thenneutralized with 1 M NaOH for 30 minutes, washed and then analyzed byXPS.

The resulting spectra are shown in FIG. 2( c). The left-most spectra inFIG. 2( c) compare the O 1 s regions for activated (upper spectrum) andcontrol chitosan films. The spectra in the O 1 s region is fit to 3peaks corresponding to C—O at ˜532.5 eV, C═O at ˜530.8 eV andphysisorbed H₂O at ˜535.3 eV. The anodically activated chitosan film hadan increased C═O peak relative to C—O peak when compared to the controlfilm, which is consistent with oxidation of the chitosan film togenerate aldehyde substituents. The middle spectra in FIG. 2( c) showthe C 1 s region which is fitted to three peaks at 287.9, 286.2, and284.6 eV corresponding to C═O/O—C—O, C—O/C—N and C—H/C—C, respectively.The C 1 s spectra show an increase in the ratio of C═O/O—C—O to C—O/C—Npeaks for the oxidized chitosan, which is consistent with results fromthe O 1 s spectra. The right-most spectra in FIG. 2( c) are for the N 1s region which is fitted to two peaks with a fixed separation of 1 eV at399.2 eV and 400.2 eV, corresponding to the amine and amide N ofchitosan, respectively. The spectra for the oxidized and control filmswere qualitatively similar in the N 1 s region.

The observed spectral differences in FIG. 2( c) were consistent with twochitosan oxidation mechanisms: (i) cleavage of primary amine and C2-C3linkage of glucosamine residues to generate a residue with two aldehydesubstituents, and (ii) oxidation at the C6 primary hydroxyl to yieldaldehyde and carboxylate functionalites. While differences in XPSspectra cannot discern between these two oxidation mechanisms (oralternative mechanisms), they are consistent with chemical changes tothe polysaccharide structure and the generation of carbonyl residues(aldehydes).

Controlled assembly of proteins onto electrodeposited andelectroactivated chitosan films in accordance with the invention can becarried out in any suitable manner. In an illustrative embodiment, achip of a type shown in FIG. 3( a) is used for the controlled assemblyof proteins onto electrodeposited and electroactivated chitosan films.This chip possessed 6 electrically independent gold electrode addresses(250 μm wide gold lines spaced 250 μm apart). Chitosan was firstelectrodeposited onto these electrode addresses using the conditionsdescribed above, rinsed, and then immersed in the activation solution(0.1 M phosphate buffer with 0.1 M NaCl). To electrochemically activatethe chitosan film at an individual address, the underlying goldelectrode was biased (0.9 V) to serve as the working electrode, while aPt wire was used as the counter electrode and Ag/AgCl was used as thereference electrode. The extent of film activation at each electrodeaddress was varied by varying the time that the anodic voltage wasapplied. The entire chip remained immersed in the activation solutionduring the sequence of film-activation steps, and the process ofactivating all 6 electrode addresses took approximately 2 minutes.

Two proteins important for biosensing applications were used todemonstrate controlled assembly onto the electrochemically activatedchitosan films. As indicated in FIG. 3( b), avidin was assembled byimmersing the chip with the activated chitosan films into a solution (4ml) containing avidin (0.15 μM) for 1 hr. After assembling the avidin,the chip was rinsed and then immersed in a solution (4 ml) containingfluorescently-labeled biotin (0.3 μM) for 0.5 hr. The fluorescencephotomicrograph in FIG. 3( b) shows a progressive increase in thefluorescence for the set of electrode addresses.

The second protein assembled onto the electrochemically activatedchitosan films was the streptococcal Fc-binding protein, Protein G. Theconditions described above were used to electrodeposit andelectrochemically activate the chitosan films at the individualelectrode addresses. Protein G was assembled by immersing the chip in 4ml of PBS solution containing protein G (0.8 μM) for 1 hr. Afterrinsing, the chip was incubated with fluorescently-labeled human IgG(0.13 μM) for 1 hr. Again, a progressive increase in intensity isapparent from the fluorescence photomicrograph in FIG. 3( c).

Next, the quantitative relationship between protein assembly andelectrochemical film activation was established. Protein assembly wasquantified using Image-J software (http://rsb.info.nih.gov/ij/) toassess the fluorescence intensities for the images in FIGS. 3( b) and3(c). The quantitative measure of electrochemical film activation is theamount of charge transferred (Q; C/m²) during anodic activation of theindividual electrode addresses:Q=∫i dtwhere i is the current density applied over time t. The plots in FIG. 3(d) show a monotonic increase in fluorescence intensity as a function ofthe charge transfer for both avidin and Protein G assembly. Theseresults demonstrate that electrochemical activation provides a simpleand rapid means to controllably assemble proteins at individualelectrode addresses. Importantly, the observed fluorescence in FIG. 3 isa measure of the proteins' functional activity (i.e., to bind eitherbiotin or IgGs).

Although the mechanism of anodic activation of chitosan has not beendefinitively established, the working hypothesis is that a reactivemediator (possibly hypochlorite OCl⁻) is electrochemically generated atthe anode, and this mediator reacts with the chitosan film to generatereactive substituents (possibly aldehydes) along chitosan's backbone.Experimentally, chitosan-coated gold wires were immersed in 1 mL ofphosphate buffer (0.1 M, pH 7.4) containing both NaCl (0.1 M) and themodel protein red fluorescent protein (RFP; 3 μM), and ananodic voltageof 0.9 V was applied for varying times. The fluorescence images andimage analysis in FIG. 12( a) show a progressive increase influorescence with anodic activation time suggesting RFP can becontrollably assembled onto the chitosan-coated gold wire. Importantly,the observed fluorescence indicates that RFP's native structure ispreserved upon electrochemical conjugation, which indicates that thisprotein assembly approach is sufficiently mild to prevent (or limit)protein denaturation.

A quantitative analysis of electrochemical RFP conjugation to thechitosan-coated wire is shown in FIG. 12( b). FIG. 12( b) indicates atrend of increasing fluorescence with charge transfer, which has beenpreviously observed with planar electrodes. (Shi, X. -W., et al.Adv.Mater. 2009, 21, 984-988.) It should be noted that the correlationbetween fluorescence and Q is considerably less scattered for the caseof planar electrodes than for the gold wires in FIG. 12( b), presumablybecause of the ease of focusing on planar surfaces during microscopicimaging.

In addition to quantitatively assembling a single protein at theelectrode addresses, the quantitative assembly of two different proteinsat the electrode addresses was demonstrated. As illustrated in theschematic representation in FIG. 4( a), two model proteins GreenFluorescent Protein (GFP) and Red Fluorescent Protein (RFP) wereassembled. Chitosan was first electrodeposited on all 6 addresses of thechip in FIG. 3( a). Next, the chitosan films on the 3 left-mostelectrodes were anodically activated by biasing them at 0.9 V forvarying times (to control Q) while the chip was immersed in a phosphatebuffer containing NaCl. After activating these three left electrodes,the chip was contacted with a PBS solution containing GFP (0.6 μM) for 1hr. After assembling GFP onto the left three electrodes, the chip waswashed with PBS containing tween (0.1%) and then immersed in a PBSsolution containing bovine serum albumin (BSA, 5%) for 2 hr to block anyresidual oxidized substituents on the chitosan film. To assemble thesecond protein, the 3 right-most electrodes were electrochemicallyactivated, and RFP was assembled using the same conditions described forGFP assembly.

The upper two images in FIG. 4( b) show fluorescence photomicrographsusing an individual green or red filter. These images indicate that GFPis selectively assembled on the left 3 electrode addresses while RFP isselectively assembled on the right 3 addresses. Little non-specificbinding is observed in these images. At the bottom of FIG. 4( b) is acomposite image using both filters and the analysis of this fluorescencephotomicrograph. FIG. 4( c) shows a plot of the fluorescence intensityvs. charge transfer (Q) for assembly of these two proteins. Sincerelatively small Q values were tested, the assembly of each protein wasnearly linear with Q. These results demonstrated the controlled assemblyof two proteins in response to localized electrical signals. Thisprotein assembly method can be extended to the sequential assembly ofmultiple proteins. Importantly, the observed fluorescence in FIG. 4indicates the native structures of RFP and GFP are retained uponassembly to the activated chitosan films.

To provide a quantitative estimate of the amount of protein assembledonto the activated chitosan films, a standard curve by spotting knownamounts of GFP and RFP onto a chitosan-coated electrode was generatedand the fluorescence was analyzed. Based on this method, it is estimatedthat protein assembly onto activated chitosan chips is on the order of10-100 pmole/cm². This level of assembly indicates that protein assemblyis comparable to, or greater than, monolayer coverage.

The invention therefore provides in various embodiments, devices withvarying numbers of proteins at varying electrode addresses. Asexemplified, a single protein may be assembled at all electrodeaddresses, or multiple proteins may be assembled at multiple electrodeaddresses. Therefore the number of types of assembled proteins rangesfrom a single protein assembled on all electrode addresses to adifferent protein assembled at each electrode.

The foregoing results demonstrate that electrode-imposed electricalsignals are usefully enlisted for the spatially-selective andquantitatively-controlled assembly of proteins. Specifically, cathodicsignals are employed for the electrodeposition of stimuli-responsiveaminopolysaccharide chitosan, while anodic signals are employed toselectively activate the chitosan film for protein conjugation. Suchon-demand protein assembly is suitably performed from aqueous solutionusing mild conditions that preserve the proteins' native structure andbiological function. This assembly approach is simple, safe andinexpensive, since no reactive reagents are required and the oxidativemediator is electrochemically-generated from NaCl. This assemblyapproach is usefully employed for the sequential assembly of proteins atindividual electrode addresses. These results further demonstrate thatchitosan possesses a unique set of properties that facilitate theintegration of biological components into electronic devices.

Electrochemical Transduction

Previous studies demonstrated that chitosan films are permeable to smallmolecules and that electrochemically active phenols (e.g., food phenols)can be anodically oxidized by chitosan-coated planar electrodes. (Wu, L.Q., et al. Adv. Funct. Mater. 2005, 15, 189-195; Wu, L. Q., et al.Adv.Funct.Mater.2006, 16, 1967-1974; Liu, Y., et al. Langmuir 2008, 24,7223-31.) To extend these observations, chitosan was electrodepositedonto gold wires (12.6 A/m² for 45 s) and these chitosan-coated wireswere used to detect the presence of the antioxidant phenol, catechin, asillustrated in FIG. 9( a) and in Example 8. It can be seen thatchitosan-coated gold wires immersed in a buffered solution containingcatechin demonstrated a strong peak at the anodic potential of 0.5 V forthe chitosan-coated wire.

Additionally, the ability of the chitosan-coated wires to performquantitative analysis was examined by the step-wise addition of aliquotsof catechin, as summarized in Example 9. Increased concentration led toincreased measured current, as illustrated in FIGS. 10( a) and 10(b).

Furthermore the effect of an increase in the number of chitosan-coatedwires was examined with regard to sensitivity of chemical detection, assummarized in Example 10. The results of Example 10, as illustrated inFIG. 10( c) indicate that the sensitivity of electrochemical detectioncan be enhanced by simply increasing the total electrode surface area ofthe chitosan-coated wires (e.g., increasing the number or increasing thelength) used for analysis.

One of the advantages of chitosan-coated electrodes for phenol detectionis the opportunity for bimodal sensing (electrical plus optical).Specifically, anodically oxidized phenols are reactive and rapidly reactwith the chitosan film to alter the film's optical properties. (Wu, L.Q., et al. Adv. Funct. Mater. 2005, 15, 189-195; Liu, Y., et al.Langmuir 2008, 24, 7223-31.) Previous studies with chitosan coatedplanar electrodes demonstrated that the optical signal (UV-visibleabsorbance) is linearly correlated with the electrical signal (chargetransferred, Q). Thus, optical signals provide an additional observablethat adds redundancy to detection. For the case of catechin, anodicoxidation products react with chitosan to generate a fluorescentproduct. (Wu, L. Q., et al. Adv. Funct. Mater. 2005, 15, 189-195.)Demonstration of this behavior for the wire format was provided by theexperiment summarized as Example 11 below.

The previous results indicate that chitosan electrodeposition andelectrochemical conjugation allow proteins to be simply and rapidlyassembled on to conducting wires. In Example 12 the ability of thesystem or device of the invention to transduce enzyme-substraterecognition into an electrical signal was examined by assembly of anenzyme on the chitosan-coated gold wires. It was demonstrated thatbiofunctionalized chitosan-coated wires can transduce biorecognitioninto electrical signals.

Specifically, the common biosensing enzyme glucose oxidase (GOx) wasused as the model for biologically based electrochemical transduction.GOx catalyzes the reaction (Lu, Y., et al. Bioelectrochemistry 2007, 71,211-6; Du, Y., et al. Bioelectrochemistry 2007, 70, 342-7; Zhou, Q., etal. J. Phys. Chem. 2007, 111, 11276-84):glucose+O₂→gluconic acid+H₂O₂The hydrogen peroxide generated by this enzymatic reaction can beanodically oxidized to generate an electrical signal by the reaction(Xi, F., et al. Biosens. Bioelectron. 2008, 24, 29-34):H₂O₂→O₂+2H⁺+2e⁻

As set forth in Example 12, two different methods were examined forassembling GOx onto the gold wire. The first approach, pioneered by Chenet al. for GOx assembly onto planar electrodes is to codeposit GOx withthe chitosan (Luo, X. L., et al. Anal. Biochem. 2004, 334, 284-289; Bai,Y. H., et al. Electrochem. Commun. 2007, 9, 2611-2616; Luo, X. L., etal. Biosens.Bioelectron.2005, 21, 190-196.). The second assemblyapproach was the electrochemical conjugation of GOx to chitosan-coatedwires.

Results shown in Example 12 with the GOx-functionalized chitosan-coatedwires demonstrate that the biological generation of electrochemicallyactive species can be quantitatively transduced into convenientelectrical signals. Consistent with other “first generation” GOx basedglucose sensors, the chitosan-coated wire offers submillimolarlimits-of-detection, while analysis of the data in FIG. 14 indicates anapparent Km of 10 mM, which is also consistent with GOx-based biosensors(Kang, X. H., et al. Anal.Biochem.2007, 369, 71-79; Manesh, K. M., etal. Biosens.Bioelectron.2008, 23, 771-779; Tan, X. C., et al.Anal.Bioanal.Chem.2005, 381, 500-507; Wu, B. Y., et al.Biosens.Bioelectron.2007, 22, 838-844; Zou, Y. J., et al.Biosens.Bio-electron.2008, 23, 1010-1016.). Additional results withGOx-functionalized wires for biosensing are provided as SupportingInformation, which can be accessed at hyper text transfer protocolinternet address: pubs.acs.org. In order to standardize fabricationmethods to ensure wire-to-wire reproducibility and to overcome problemsof interference mediators have been used in later generations ofGOx-based glucose sensors (Castillo, J., et al. Sens. Actuators, 2004,102, 179-194; Heller, A., et al. Chem.Rev.2008, 108, 2482-2505; Wang,J., et al. Chem.Rev.2008, 108, 814-825.).

It is shown herein that chitosan is a unique material that “recognizes”cathodic signals and responds by electrodepositing as a stable film.Further, anodic signals can activate chitosan for protein assembly, thusallowing chitosan to be biofunctionalized. These capabilities ofchitosan facilitate the coupling of the molecular recognition propertiesof proteins (e.g., glucose oxidase) with the transduction capabilitiesof metals for electrical signaling. Importantly, electrodeposition andelectrochemical protein conjugation are achieved rapidly without theneed for reactive reagents or complex activation and protection steps.Thus, the preparation of biofunctionalized chitosan-coated wires issimple, safe, and inexpensive.

Chitosan-coated wires are an interesting platform that can be viewed aseither conducting fibers or functionalized wires. This platform isparticularly attractive for biosensing applications outside thelaboratory for two general reasons: First, electrochemical detection canbe performed in simple, robust, and inexpensive systems that can beminiaturized as hand held or wearable devices; and second, fibers (orwires) of different functionalities can be prepared and assembled on a“mix-and-match” basis to tailor the biosensing capabilities to thespecific needs. Thus, assemblies of chitosan-coated wires may allowmultiplexed biosensing in the field while accessing the power ofelectronics for signal processing and wireless communication.

In sum, the above results demonstrate that chitosan-coated wires cantransduce chemical information of their environment into convenientelectrical signals. As demonstrated in Examples 9-11, thechitosan-coated wires provide a convenient electrical signal fordetection and quantification. Using the exemplary protein catechin, thechitosan-coated wire provided an additional optical (i.e., fluorescence)signal that can be used to provide confirmatory information. It shouldbe noted that while the electrical signal is an instantaneous measure,the fluorescence is a cumulative measure of protein.

Devices of the invention may be used in formation of filaments or fibersor in the generation of textiles or garments comprising such devices.The devices of the invention may be utilized as wearable instruments orsmart textiles. In one embodiment the wearable instruments or smarttextiles are utilized as a sensor to detect condition of the wearer oruser. Conditions sensed by the sensor may include, but are not limitedto a condition of wearer such as vital signs, temperature, heart rate,blood pressure, respiration rate, etc. and/or ambient conditions such astemperature, pressure, light, etc. or other environmental stimuli, suchas those from mechanical, thermal, chemical, electrical, magnetic orother sources. Smart textiles are capable of a range of functions, suchas sensing, detecting, initiating, reacting, regulating andcommunicating.

In another embodiment the devices of the invention are used in hand-helddevices for applications such as point-of-care detection and diagnosisof disease, assessment of environmental samples, and food safetyassessment.

The advantages and features of the invention are further illustratedwith reference to the following examples, which are not to be construedas in any way limiting the scope of the invention but rather asillustrative of specific embodiments of the invention in particularapplications thereof.

The following examples use materials obtained as follows:

The following materials were purchased from Sigma-Aldrich: chitosan fromcrab shells (85% deacetylation and 200 kDa, as reported by thesupplier); atto 565 labeled biotin; human IgG labeled with fluorescein;bovine serum albumin (BSA); Tween 20; Goldwire (99.9+%, 0.25 mmdiameter), (+)-catechinhydrate, D-(+)-glucose (99.5%), and glucoseoxidase (GOx from Aspergillusniger; 136 kU/gm).

Avidin D was purchased from Vector Laboratories. Texas Red labeled BSAwas purchased from Invitrogen.

Platinum/silver wires (99.95%) were purchased from Surepure ChemetalsInc.

NHS-Fluorescein was purchased from Pierce.

Nonfat dry milk was purchased from Lab Scientific.

Red fluorescent protein (RFP), green fluorescent protein (GFP) andProtein G were expressed from E. coli and purified using standardmethods (X W Shi, et al, Biomacromolecules 2008, 9, 14-17). Siliconwafers were patterned using standard photolithographic methods (L -Q.Wu, et al, Langmuir 2003, 19, 519).

EXAMPLE 1 Chitosan Electrodeposition

A chitosan solution (0.9 w/v %) was used for chitosan electrodeposition.This solution was prepared by adding chitosan to 1% HCl, mixingovernight, and filtering using a vacuum filter to remove undissolvedparticles. The pH of this chitosan solution was then adjusted to 5.6using 1 M NaOH.

Chitosan films were electrodeposited on two adjacent electrodes of thechip in FIG. 2( b). Chemical analysis of electrochemically-activatedchitosan films was performed using X-ray Photoelectron Spectroscopy(XPS). One film served as the control, while the other was anodicallyactivated. XPS analysis was performed on a Kratos Axis 165 using Al Ka(1486.7 eV) radiation at 300 W(http://www.chem.umd.edu/facility/xps.php). The system was operated inthe hybrid mode, survey spectra were collected with a pass energy of 160eV and high resolution spectra at a pass energy of 20 eV. The workingpressure of the instrument was at 1×10⁻⁸ Torr or better throughout datacollection. All spectra were calibrated to the hydrocarbon peak at 284.6eV. Peak fitting was carried out after the application of a Shirleybackground, peaks with a 60% Gaussian, 40% Lorentzian line shape wereused to fit the O 1 s, C 1 s and N 1 s regions with FWHM values equal to1.90, 1.50, and 1.75 eV respectively.

EXAMPLE 2 Protein Assembly

Protein assembly was performed using three steps. First, chitosan waselectrodeposited by partially immersing the patterned chip into thechitosan solution (0.9%, pH 5.6), applying a cathodic voltage to achievea constant current density of 4 A/m² for 15 sec. After deposition, thechips were rinsed with water. The second step was to electrochemicallyactivate the deposited chitosan film. Activation was performed bypartially immersing the chips in a 0.1 M phosphate buffer (pH 7)containing 0.1 M NaCl, connecting the chitosan-coated electrode to serveas the working electrode in a 3-electrode system, and biasing thechitosan-coated electrode to an anodic voltage of 0.9 V to achieve aspecific charge transfer (Q). The three-electrode system (CHI627Celectrochemical analyzer, CH Instruments, Inc.) employed Ag/AgCl as thereference electrode and a Pt wire as the counter electrode. For thethird step, protein conjugation to the activated films, the chips werewashed with 0.1 M PBS, and then immersed in a solution containing thetarget protein and incubated for 1 hr (no power was supplied during thisthird step). Protein assembly was observed using fluorescence microscopy(Leica MZFL III) and the images were analyzed using ImageJ software(http://rsb.info.nih.gov/ij/).

EXAMPLE 3 NaCl is Required for Anodic Activation

FIG. 5 compares protein binding to chitosan films activated in 0.1 Mphosphate buffer (pH=7.0) with and without NaCl. Initially, chitosanfilms were electrodeposited on both electrodes of the patterned chipshown in FIG. 5( a). After washing with water, the right electrode wasconnected to a power supply and served as the working electrode in a3-electrode system (Ag/AgCl as a reference and Pt wire as a counterelectrode) and the electrodes were immersed in 0.1 M phosphate bufferwith 0.1 M NaCl. In this example, a somewhat different procedure wasused to activate the chitosan film. Specifically, the potential of theunderlying electrode was linearly increased from 0 to 1.5 V at rate of100 mV-s⁻¹. This linear increase in voltage was repeated a total of 5times. The chitosan film on the left electrode served as a control inwhich the same “activation” procedure was performed in 0.1 M phosphatebuffer lacking NaCl. After washing with 0.1 M PBS buffer for 20 min, thechip was immersed in a solution containing red fluorescent protein (RFP;0.6 μM) for 1 hr. FIG. 5( b) shows the fluorescence photomicrograph froma Leica fluorescence microscope (MZFL III) and analysis by Image Jsoftware (http://rsb.info.nih.gov/ij/). Substantial fluorescence wasobserved on the right electrode on which the chitosan film had beenanodically oxidized in the presence of NaCl, while little fluorescencewas observed on control chitosan film on the left electrode. Theseresults indicate that anodic activation of chitosan requires thepresence of NaCl and suggests that oxidized NaCl species (e.g., HOCl orOCl⁻) may serve as the mediator for film activation.

EXAMPLE 4 Chitosan is Required for Electrochemical Protein Assembly

FIG. 6 compares protein assembly to an activated chitosan film withassembly to an uncoated (bare) gold electrode. In this example, the chipin FIG. 5( a) was used, chitosan was electrodeposited on the leftelectrode, and the right electrode was uncoated. During anodicactivation, both electrodes were biased to an anodic potential (0.9 V)and the charge transfer was 80 C/m². After washing the chip with PBSbuffer for 20 minutes, the chip was immersed in fluorescently-labeledbovine serum albumin (Texas red-BSA; 0.5 μM) for 1 hr. After washing thechip with PBS containing 0.1% Tween, the chip was examined usingfluorescence microscopy. The images in FIG. 6 show red fluorescence onthe chitosan film on the left electrode while no fluorescence wasobserved on the gold electrode that lacked the chitosan film. Thisresult indicates that anodic treatment activates the chitosan film forsubsequent protein assembly, while the gold electrode was not activatedby biasing to anodic conditions.

EXAMPLE 5 Oxidized Substituents of Chitosan are Responsible for ProteinAssembly

FIG. 7 compares protein assembly to an activated chitosan film, with andwithout treatment with a chemical reducing agent. In this example,chitosan was electrodeposited to both electrodes of the chip shown inFIG. 5( a). The chitosan film on the right electrode was activated bybiasing the underlying electrode to an anodic potential (0.9 V to acharge transfer of 80 C/m²). Next, the chip was contacted with thereducing agent NaBH₄ (0.2 mg/ml) for 1 hr. After washing the chip withPBS, the chitosan on the left electrode was anodically activated bybiasing its underlying electrode (0.9 V, Q=80 C/m²). After washing thechip with PBS buffer for 20 minutes, the chip was contacted with asolution of the labeled BSA (0.5 μM) for 1 hr. FIG. 7 shows strongfluorescence on the left chitosan film, while low levels of fluorescencewere observed on the right film. This result indicated that theactivated chitosan film was “de-activated” by treatment with a reducingagent, and that anodic activation leads to oxidation of the chitosan,and the oxidized substituents (aldehydes) are capable of reacting withproteins.

EXAMPLE 6 Second Example of Chitosan Electrodeposition

A chitosan solution (1 w/w %) was prepared by adding chitosan flakes towater and slowly adding 1% HCl to dissolve the polysaccharide (final pH5.6). After mixing overnight, the solution is filtered using a vacuumfilter to remove undissolved particles. Fluorescein-labeled chitosan wasprepared from a chitosan film. The film was prepared by pouring achitosan solution into a Petri dish, drying overnight at 45° C.,neutralizing the film with 0.1 M NaOH for 30 min, and then washing thefilm with water. The film was labeled by placing it in 10 mL of water,adding NHS-fluorescein (5 mg/mL in DMSO) to a final concentration of 0.5μg/mL, and incubating at room temperature for 30 min. After labeling,the labeled chitosan film was washed with copious amounts of water. Thelabeled chitosan film was then dissolved by adding small additions of 1%HCl to achieve a pH between 5 and 6.

Electrodeposition and codeposition were performed using 1 cm gold wires.Before use, the gold wires were cleaned by immersing in piranha solution(7:3 concentrated H₂SO₄:30%H₂O₂) for 30 min. For deposition, the goldwires were immersed in a solution of chitosan (for code positionstudies, the chitosan solution also contained 680 U/mLGOx). An alligatorclip was used to connect the wire to a DC power supply (2400Sourcemeter, Keithley) and the gold wire was biased to serve as thecathode (negative electrode) while a platinum wire served as anode.Electrodeposition was performed at a constant current density of 12.6A/m² for 15-90 s (typical voltages of 2-3 V). After electrodeposition,the wire was immediately removed from the deposition solution, brieflyrinsed with water, and dried in air before use.

EXAMPLE 7 Second Example of Protein Assembly

Electrochemical protein conjugation to chitosan-coated wire was achievedusing a procedure adapted from a previous study (Shi, X. W., et al.Adv.Mater. 2009, 21, 984-988.). First, chitosan was electrodeposited byimmersing 1 cm of gold wire into the chitosan solution and applying acathodic voltage at a constant current density of 12.6 A/m² for 20 s.After deposition, the wires were rinsed with water. The second step wasto electrochemically conjugate protein (either RFP or GOx). Conjugationwas performed by (i) immersing the chitosan-coated wire in a solution(0.1 M phosphate buffer with 0.1 M NaCl, pH 7.4) containing protein,(ii) connecting the chitosan-coated electrode to serve as the workingelectrode in a three-electrode system, and (iii) biasing thechitosan-coated electrode to ananodic voltage of 0.9 V (vs Ag/AgCl) fora specific time (typically 1 min). The protein-functionalized wires werethen washed three times (10 min each) in 0.1 MPBS (containing 0.05%Tween 20) to remove nonspecifically bound protein.

The wires were examined using a Leica fluorescence microscope (MZFLIII).To observe fluorescence associated with fluorescein-labeled chitosan, aGFP plus filter with excitation filter at 480/40 nm and emission filterat 510 was used. The fluorescence associated with the catechin-modifiedchitosan was observed using a GFP filter with an excitation filter at425/60 nm and an emission filter at 480 was used. To observe redfluorescence (e.g., for RFP), a Leica 41004 TXRD filter was used withexcitation filter at 560/55 nm and an emission barrier at 645/75 nm.Fluorescence micrographs were obtained using a digital camera (spot 32,Diagnostic Instrument) connected to the fluorescence microscope and theimages were analyzed using ImageJ software (hyper text transfer protocolinternet address: //rsb.info.nih.gov/ij/).

EXAMPLE 8 Detection of the Presence of Catechin

A chitosan-coated wire was immersed in a buffered solution containingcatechin (2 mM, pH 7.4) and a cyclic voltammogram (CV) was generated.FIG. 9( b) shows a strong peak at the anodic potential of 0.5 V for thechitosan-coated wire. A CV for a control chitosan-coated wire immersedin a catechin-free solution shows no anodic peak. An uncoated gold wireimmersed in the catechin-containing solution was tested as a secondcontrol. The CV for this uncoated wire also shows a peak current near0.5 V, consistent with catechin's oxidation; however, the peak currentfor this control is considerably less than that observed for thechitosan-coated wire. The higher sensitivity of chitosan-coatedelectrodes has been previously observed and is presumably due to apreconcentration effect (Liu, Y., et al. Langmuir 2008, 24, 7223-31.).

Electrochemical measurements such as cyclic voltammetry (CV),amperometric current versus time measurements, and chronocoulometry werecarried out with a CHI627C Electrochemical Analyzer (CH Instruments,Inc., Austin, Tex.). Measurements were performed using a standardthree-electrode configuration and the specific experimental conditionsprovided herein.

EXAMPLE 9 Quantitative Analysis of Phenol Levels

Chitosan-coated wires were immersed in a buffered solution (0.1 Mphosphate, pH 7.4), biased to 0.5 V, and aliquots of catechin weresequentially added to increase the concentration by 0.6 μM, asillustrated in FIG. 10( a). The inset to FIG. 10( b) shows that aftereach catechin addition, a step-change in current is observed. The plotin FIG. 10( b) shows a strong correlation between the current and thecatechin level. The micromolar limit of detection observed in FIG. 10(b) is considerably larger than the typical catechin concentrations foundin wines and tea. (Dalluge, J. J., et al. J.Chromatogr.,2000, 881,411-424; Arts, I.C.W., et al. J. Agric. Food Chem. 2000, 48, 1752-1757.)

EXAMPLE 10 Sensitivity of Electrochemical Detection

In Example 10 it was examined how the sensitivity of electrochemicaldetection could be increased by simply increasing the number ofchitosan-coated wires used for detection (i.e., by increasing theelectrode surface area).

The inset to FIG. 10( c) shows the step increase in current observed foreach catechin addition for systems containing one, three, or fivechitosan coated wires. The plot in FIG. 10( c) shows an early linearcorrelation between the observed current and the catechin concentrationfor each of these experimental systems. As expected, the slopes of theseplots (i.e., the sensitivity) increase in the order 0.023, 0.062, and0.17 μA/μM for the one-, three-, and five-wire systems, respectively.

EXAMPLE 11 Fluorescent Detection of Catechin

Chitosan-coated gold wires were immersed in solutions containingdiffering concentrations of catechin and applied a positive (i.e.,anodic) voltage of 0.5 V for 2 s. After this reaction, the wires wererinsed and examined using fluorescence microscopy. FIG. 11 shows aprogressive increase in fluorescence of the chitosan-coated wires withthe catechin levels in the solution. Two controls are shown in FIG. 11.The first control, shown at the left, is for a chitosan-coated wireimmersed in a catechin solution (1 mM) for 2 s without applying avoltage. The second control, shown at the right in FIG. 11, is for achitosan-coated wire immersed in a solution lacking catechin, but biasedto 0.5 V for 2 s. Neither control displays significant fluorescence.Also, an uncoated gold wire does not become fluorescent upon catechinoxidation under the same conditions (data not shown).

EXAMPLE 12 Electrochemical Transduction

Codeposition of GOx

GOx is mixed into the chitosan-containing solution and electrodepositionis performed using this GOx chitosan solution. The electrodepositedchitosan film appears to have a sufficiently fine mesh-size that thecodeposited proteins are entrapped within the film's network for sometime. GOx was codeposited from a solution (680 U/mL GOx, 1% chitosan,pH5.6) for 20 s at a current density of 12.6 A/m². After codeposition,the biofunctionalized wire was immersed in a solution containing glucose(2 mM) and a cyclic voltammogram (CV) was generated. FIG. 13( a) shows astrong anodic signal for the wire with codeposited GOx. Two controlswere performed: a chitosan-coated wire lacking GOx that was immersed inthe glucose-containing containing solution and a GOx-chitosan-coatedwire immersed in a solution lacking glucose. The CVs for these controlsshow no anodic peaks.

Electrochemical Conjugation of GOx

Chitosan was first electrodeposited onto the wire under cathodicconditions. Then the chitosan-coated wire was immersed in a solutioncontaining buffer (0.1 M phosphate, pH 7.4), NaCl (0.1 M), and GOx (680U/mL), and the wire was biased to 0.9 V for 60 s. After electrochemicalconjugation of GOx, the biofunctionalized wire was immersed in theglucose solution (2 mM) and a CV was generated. FIG. 13( b) shows astrong anodic signal for this electrochemically conjugated wire. As acontrol, a chitosan-coated wire was conjugated with milk by performingelectrochemical conjugation in a solution containing nonfat dry milk(5%). The CV for this milk-conjugated control wire in the 2 mM glucosesolution shows no anodic signal. These initial results indicate that (i)GOx can be assembled onto (or within) the chitosan-coated wire, (ii) GOxretains its biological (i.e., catalytic) function, and (iii) GOxfunctionalized wires can transduce information of its environment (i.e.,the presence of glucose) into electrical signals.

Steady State Current vs. Glucose Concentration

Finally, a standard curve between the steady state current and theglucose concentration for GOx functionalized chitosan-coated wires wasgenerated. When methods described above were used, cathodic signals wereused to electrodeposit chitosan onto the gold wire and then anodicsignals were used to conjugate GOx to the chitosan-coated wires. TheseGOx-functionalized wires were then immersed in a buffer (0.1 Mphosphate, pH 7.4) and biased to 0.8 V to serve as the working electrodein the three-electrode system. Analogous to the studies with catechin(FIG. 10), aliquots of glucose were then added to the buffer (eachaliquot increased the glucose concentration by 0.25 mM). The inset inFIG. 14( a) shows a step-increase in anodic current was observed aftereach glucose addition, while the standard curve shows an early linearrelationship between anodic current and glucose concentration. In aseparate experiment with a different GOx-functionalized chitosan-coatedwire, FIG. 14( b) shows that a similar relationship was observed over abroader range of glucose concentrations.

The foregoing examples demonstrate the utility of the invention for useof electrical signals to effect protein assembly.

While the invention has been has been described herein in reference tospecific aspects, features and illustrative embodiments of theinvention, it will be appreciated that the utility of the invention isnot thus limited, but rather extends to and encompasses numerous othervariations, modifications and alternative embodiments, as will suggestthemselves to those of ordinary skill in the field of the presentinvention, based on the disclosure herein. Correspondingly, theinvention as hereinafter claimed is intended to be broadly construed andinterpreted, as including all such variations, modifications andalternative embodiments, within its spirit and scope.

What is claimed is:
 1. A method of forming a bioelectronic deviceincluding a protein on an electrically conductive substrate, comprising:electrodepositing aminopolysaccharide chitosan on the substrate whileapplying a cathodic voltage to the substrate, to form anaminopolysaccharide chitosan film thereon; applying an anodic voltage tothe substrate in the presence of NaCl to activate theaminopolysaccharide chitosan film so that it is reactive with protein;and reacting the aminopolysaccharide film, after activation thereof,with the protein, so that the protein assembles on and is coupled to thesubstrate, thereby forming said bioelectronic device.
 2. The method ofclaim 1, wherein the electrically conductive substrate comprises anelectrode on a silicon chip.
 3. The method of claim 2, wherein theelectrode comprises a gold electrode or a copper electrode.
 4. Themethod of claim 1, wherein the protein is selected from the groupconsisting of avidin, an immunoglobulin-binding protein, protein G, redfluorescent protein, green fluorescent protein, and glucose oxidase. 5.The method of claim 1, wherein the substrate comprises a multiplexingchip having a multiplicity of electrode addresses, wherein the electrodeaddresses independently have said protein assembled thereon and coupledthereto.
 6. The method of claim 5, wherein said protein comprises atleast one biosensor protein species.
 7. The method of claim 5, whereinthe electrode addresses have different protein species assembled thereonand coupled thereto.
 8. The method of claim 7, wherein said differentprotein species comprise biosensor protein species.
 9. The method ofclaim 7, wherein the different protein species are selected from avidin,an immunoglobulin-binding protein, protein G, red fluorescent protein,green fluorescent protein and glucose oxidase.
 10. A method of formingan electrically conductive substrate adapted for assembly of a proteinspecies thereon, said method comprising: electrodepositingaminopolysaccharide chitosan on the substrate while applying a cathodicvoltage to the substrate sufficient to form an aminopolysaccharidechitosan film thereon; and applying an anodic voltage to the substratein the presence of NaCl to activate the aminopolysaccharide chitosanfilm so that it is reactive with the protein species.